The Idl &ld3 genes and proteins are required for tumor-associated angiogenesis. We hypothesize that inhibiting the activity of the Idl and/or Id3 transcription activators will retard the growth & metastases of tumors. However, the effort to discover an orally active, small molecule, anti-cancer drug by identifying an molecule that inhibits the action of the Idl & Id3 proteins requires bioanalytical methods to screen for small molecules that inhibit the binding of Idl & Id3 to their natural ligands, the E-proteins, and also to establish which cancers would be most likely to respond to an anti-ld treatment. Such bioanalytical method are not currently available for the Id proteins, but we recently obtained promising preliminary results by monitoring the extent of the interaction of horseradish peroxidase-labeled E47 with Idl captured by an anti-ldl antibody bound to microtiter plates. This procedure resembles a traditional ELISA, but uses a natural protein ligand (E47) to the analyte (Idl) instead of a second antibody to detect capture. When fully developed and validated, this method will be used to both measure Idl & Id3 in the blood of patients with certain advanced cancers to determine whether it has potential as a diagnostic method, and also to evaluate the ability of small molecule inhibitors to reduce Id activity. To advance this effort, we plan to (1) validate the pilot assay with respect to limit of quantitation, precision, accuracy, reproducibility, repeatability and stability, (2) use the validated assay to determine if Id concentrations can be reliably measured in the serum and tissue from different mouse models of human cancer, and (3) use the validated assay, configured with fixed & physiologically relevant concentrations of Idl and E47 to explore disruption of Idl binding to E47 at various concentrations of candidate small molecule inhibitors of binding. It is expected that completion of the research described in this grant will result in an assay useful for high throughput screening of chemical libraries for anti-ld activity, and in the selection of those cancers, and perhaps stages of cancer, where anti- Id treatment is most likely to be effective. The assay would, at a minimum, have the potential to be developed into a practical diagnostic/prognostic product to select patients for clinical trials and to monitor their response to anti-ld therapy. It is also possible that the assay could be a commercial product depending on the technical and medical success achieved.